The overall goal of this work is to develop anatomical, functional, and molecular magnetic resonance imaging (MRI) techniques that allow non-invasive assessment of brain function and apply these tools to study plasticity and learning in the rodent brain. MRI techniques are having a broad impact on understanding brain. Anatomical based MRI has been very useful for separating gray and white matter and detecting numerous brain disorders. Functional MRI techniques enable detection of regions of the brain that are active during a task. Molecular MRI is an emerging area, whose major goal is to image a large variety of processes in tissues. The goal of this project is to translate MRI developments in all these areas to study system level changes that occur in the rodent brain during plasticity and learning. Aim 1: Over the past few years, we have completed studies in the rodent brain that acquired very high temporal and spatial resolution functional MRI (fMRI) to monitor changes in hemodynamics as a surrogate marker of electrical activity during forepaw stimulation. Over the past year we have demonstrated that fMRI from single venous vessels from deep cortex can be effectively imaged. fMRI has long been able to detect individual draining vessels but now we have demonstrated the ability to detect individual vessels in deep tissue. Ongoing studies are aimed at detecting individual arterioles to complement our detection of individual venuoles. Work over the past year has also been completed that is aimed at obtaining very high spatial and temporal resolution fMRI images to determine if the onset of fMRI signal corresponds to the input layer for neural signaling. A one dimensional imaging technique enables us to achieve 50 micron spatial resolution through the cortex and 50 msec temporal resolution. In somatosensory cortex, fMRI signals start in layer 4 at about 600-800 msec consistent with our previous work. In motor cortex fMRI onset corresponds to the neural input in mid-cortical areas. In a model where neural input into somatosensory cortex switches to beginning in layer 2/3 and or layer 5 rather than layer 4, the fMRI onset also switches to layer 2/3 and layer 5. This exciting preliminary work is consistent with the hypothesis that the onset of fMRI enables extracting information about the onset of neural activity in a brain region. In the coming year we will verify this idea as well as consider beginning studies on the human brain to look at onset dynamics at high spatial temporal resolution. Aim 2: Over the past several years we have demonstrated that manganese chloride enables MRI contrast that defines neural architecture, can monitor activity, and can be used to trace neural connections. Over the last couple of years we have completed the assignment of cortical layers detected using manganese enhanced MRI by comparison to histology and have demonstrated that functional anatomy of several cortical regions of the rodent brain can be defined in individual animals. In particular, clear cytoarchitectural boundaries can be detected between numerous brain areas enabling, for the first time, cytoarchitectural changes to be followed in individual brains over time. In addition, we have completed studies that trace the laminar inputs of the olfactory pathway from the olfactory bulb to rodent frontal cortex. We have used this laminar specific tracing to determine whether manganese enhanced MRI can detect changes due to learning. In a simple fear conditioning experiment (odor with foot shock)a small increase in manganese influx from olfactory cortex to orbital frontal cortex was the only significant change detected. Analysing this change at higher spatial resolution indicated that tracing of manganese was increased by 50% into layer 1 of orbital frontal cortex. This predicts a strengthening of this synapse. There were increases into sub-regions of the amygdala as well In the coming year we will use this information to guide further study of synaptic strength changes during fear conditioning under Aim 4. The ability to detect cytoarchitecture with MRI has led us to apply MRI to analysing an mouse model of Alzheimers that has neurodegeneneration of olfactory neurons. MRI results clearly demonstrate loss of the enhancement in the glomerular layer of the olfactory bulb, clearly proving the usefulness of laminar specific MRI for studying disease processes. Aim 3: Over the past few years we established a rodent model that uses peripheral denervation to study brain platicity in response to the injury. Over the past couple of years we have showed that denervation of the infraorbital nerve leads to large increases in barrel cortex responses along the spared whisker pathway as well as large ipslateral cortical activity consistent with our previouus work in the forepaw and hindpaw. fMRI and manganese enhanced MRI predicted a strengthening of thalamo-cortical input along the spared pathway which was verified in slice electrophysiology studies in collaboration with John Isaac's laboratory. Prior to this it was widely beleived that the thalamo-cortical input was not capable of strengthening after the critical period. The mechanisms of this strengthening are under study. Interestingly, the denervation is causing a re-activation of the ability of this synapse to demonstrate long term potentiation which is usually lost after the critical period durinf the first week of life. Over the past year we have also focused on understanding the role of the ipsilateral activation that depends on neural signalling from the spared cortex to the ipsilateral cortex via the corpus callosum. The goal is to determine the significance of this long distance cortical plasticity as well as determine using MRI where the synaptic changes occur underlying this change in activity. In a series of studies we have shown that this ipsilateral activation helps to protect cortical territory from take over by adjacent cortical representations. We will continue testing the hypothesis that we will continue to test is that when one side of a body part is denervated, the spared side occupies the injuried side cortex via a corpus collasoal pathway. Furthermore evidence from Manganese based track tracing shows a strengthening of input into layer 2/3 and 5. Over the next year we will use slice electrophysiology to verify this strengthening in analogy to the work done on the talamo-cortical synapse. Aim 4: We have begun to explore the use of advanced MRI tools for studying simple learning paradigms in the rodent. In order to accomplish this we have been developing techniques that will enable routine fMRI in awake rodents. While fMRI is widely performed in humans and awake primates there have only been a few scattered studies on awake rodents. Training regimens and techniques to hold the head have been developed over the past two years. Interestingly, we have large differences in brain fMRI activation due to somatosensory stimulation or visual stimulation in the awake animal vs anesthetized animal that are stimulation dependent. Somatosensory stimulation gives a strong fMRI response in anesthetized but not awake rodents and visual stimulation give a strong response in awake but not anesthetized animals. Electrophysiology from these areas verifires this result. This is important to lay the ground work for the best types of stimuli that give good fMRI responses in the awake rodent to better design behavioral pardigms that are consistent with fMRI. We have also expanded the number of stimuli that can be used to include high frequency auditory stimuli. It is clear that rodents do some forms of communication at high frequency and these high frequencies enable us to avoid the lower frequency noise produced by the MRI scanner.